How Can Cells Divide When DNA Looks Like Spaghetti?

Topoisomerases untangle the mess, very carefully, with itty-bitty molecular scissors.

DNA is incredibly elegant as a solution to information storage and heredity. But it is also an enormous mess, with the genome of humans extending to five feet in combined length. (Imagine 9,000 miles of garden hose.) So each cell, which contains this whole amount, has a nucleus resembling an incredibly convoluted nest of spaghetti. Yet at mitosis, the chromosomes condense, separate, and neatly partition to each new cell. Some of the solution consists in how the DNA lies in the interphase cell- it is already somewhat pre-organized there. But most of the solution comes from enzymes that do not bother disentangling it- they cut the Gordian knot with enzymatic swords. Genomes were able to surmount the length problem over evolutionary time by the development of topoisomerases, which cut and religate DNA with extraordinary precision.

There are two main types of topoisomerase (named for altering topology, or the organization / twisting of DNA, without changing its energy, sequence, or composition). Topisomerase I cuts only one strand of the double-standed DNA, and can thus relieve coiling tension. Some forms can wind up the tension, using ATP. Topoisomerase II cuts both strands, and is the main enzyme that allows complete de-catenation / detangling of DNA during replication, transcription, meiosis, as well as mitosis. A recent paper looked deeply into the mechanism of this class of enzymes.

As one can imagine, the minimal requirements of a Topoisomerase II is that it hold on to both ends of the DNA it has cut, while passing through the other DNA strand which has, by virtue of general tangling, come up against it. This condition of collision needs to be detected, prior to being resolved, so that the enzyme is properly positioned. The complex also has to detect that the process has finished, and reset to the starting state, including religation of the cleaved strand of DNA. It is a tall order for a mere chemical confection to carry out, frankly.

But it turns out that enzymes can have hands, if not brains. The authors provide, on the basis of a great deal of past work as well as their own, a compelling model of how this topoisomerase performs its amazing feats.

Molecular structure of a typical topoisomerase II, composed of two copies of Top6A (green and red; second copy in gray) and two copies of Top6B (yellow, purple, and orange; second copy in gray). The G-segment DNA (~70 bp) is strung along the underside, and the T-segment DNA will shortly be accommodated in the middle, upon which the top portions clamp together. Cylinders represent alpha helices, the common secondary structure of proteins. Key domains for the activity of this protein complex are noted- the H2TH domain, which notifies Top6B that a G-segment is present- the KGRR domain, which notifies the same enzyme that a T-segment is present, and to keep the clamp closed. And lastly, the stalk/WKxY domain, which in addition to helping to bind the G-segment communicates between Top6B and Top6A that cleavage of the G-segment can happen. The G-segment will be cleaved in half at the bottom of the structure, later to be re-ligated after the T-segment has passed through. 

The enzyme (it is a tetramer made up of dimers of two separate proteins, Top6A and Top6B) forms a large hoop, with arms outstretched that will join during its action. One DNA segment, the one to be cut (the G-segment, for gate) is first bound by the underside of the hoop, centered at the middle active site which does the cutting, holding, and religation. The outstretched arms encompass the other DNA strand (the T-segment, for transit). Both top and bottom of the complex have ATPase activity, though for different purposes.

The key finding made by these authors is that the G-segment DNA is bound not only near the cleavage site (in Top 6A), but by the entire arm structure, up to a domain in Top6B that the authors call H2TH. About 70 basepairs of the G-segment DNA are bound, overall. This not only stabilizes and holds this DNA while it is being cut and the T-segment is being passed through, but it also allows the Top6B portion of the enzyme to sense the status of the whole complex, so that it can properly sequence its activities.

The KGRR feature functions to sense T-segment DNA and keep the clasp closed and ATP unhydrolyzed while the T-segment is present. The bottom graphs show ATP hydrolysis in the mutants diagrammed above, while the gel images show relaxation of the supercoiled DNA but the enzyme (moving it from the bottom to the top, left to right). ATP hydrolysis is increased to a free-wheeling state, while DNA relaxation fails to happen, in two mutant versions of the KGRR finger.

For example, the authors identify another feature near the top of Top6b, called KGRR, which is a finger that points inwards to touch the T-segment DNA. When they mutate it, they find that ATP is now freely digested in the presence of supercoiled DNA, much more actively than by the intact (wild-type) enzyme. But the enzyme is inactive ... no strand passage takes place, and supercoils are not relieved. The mutant enzyme is spinning its wheels, clasping and opening without doing anything. What this indicates is that in the normally functioning enzyme, the KGRR domain is a sensor that keeps the complex locked up till the T-segment passes out the other side, via the cleavage in the G-segment. Only then can ATP be digested by both halves of the enzyme, re-ligating the G-segment, and opening the Top6B arms to allow a new round of stress relief to take place.

Similarly, they conclude that the function of the H2TH sensor is in part to notify the Top6B part of the enzyme that a G-segment DNA is bound on the underside, allowing ATP to be bound and the clasp to close, if a T-segment also happens along. T-segments should not bind unless a G-segment is bound first. Secondly, the dramatic DNA bend adopted by the G-segment in this protein structure, especially in the locked-up conformation, draws on the supercoiling / torsional state of the DNA that is the target of action. Supercoiled DNA binds with 60-fold higher affinity than unstrained DNA.

Overall schematic of the mechanism of the enzyme- see text.

To recapitulate, the overall model is that G-segment DNA, in torsionally stressed condition, binds to the broad binding area of the underside of the enzyme. This notifies the Top6B domain that T-segment binding is acceptable. When that happens, the clasp is closed and ATP is bound, but not hydrolyzed, setting up the next step. A hinge between the two protein halves notifies Top6A that it can cut the G-segment. When that is done and the T-segment passes through, the KGRR sensor notifies the Top6B that the clasp is empty, so the ATP is hydrolyzed and the clasp releases, ready for another round.

It is an intricate molecular mouse-trap, built of ratchets and sensors of various kinds, using the jostling motion universal at this scale, plus key inputs of energy (ATP) to accomplish what on the large scale looks like an amazing feat of re-organization.

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